US10272070B2 - Method for treating neurodegenerative diseases - Google Patents

Method for treating neurodegenerative diseases Download PDF

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US10272070B2
US10272070B2 US15/518,438 US201515518438A US10272070B2 US 10272070 B2 US10272070 B2 US 10272070B2 US 201515518438 A US201515518438 A US 201515518438A US 10272070 B2 US10272070 B2 US 10272070B2
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Khoa Dinh NGUYEN
Edgar G. Engleman
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Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/18—Benzimidazoles; Hydrogenated benzimidazoles with aryl radicals directly attached in position 2
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar

Definitions

  • the present invention relates to methods for treating neurodegenerative diseases by administering to a subject a Ppargc1a activator, 2-(4-tert-butylphenyl)-1H-benzimidazole, 2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole.
  • ALS Amyotrophic lateral sclerosis
  • Activation of immune cells in the central as well as peripheral nervous system has been suggested to be a critical determinant of disease progression in ALS (Phani et al, Front Pharmacol. 3:150, 2012). Specifically, microglia and macrophages have been shown to play distinct roles in the orchestration of neuroinflammation in this disease (Dibaj et al, PLoS One. 6(3):e17910, 2011; Boillee et al, Science, 312:1389-92, 2006).
  • BMT bone marrow transplantation
  • AD Alzheimer's Disease
  • oxidative stress has been proposed to be an underlying cause of neurodegeneration in AD (Friedland-Leuner et al Mol Biol Transl Sci, 127:183-201, 2014).
  • AD Alzheimer's disease
  • Parkinson's disease also known as idiopathic or primary parkinsonism
  • PD Parkinson's disease
  • the motor symptoms of PD result from the death of dopamine-generating cells in the substantia nigra, a region of the midbrain; the cause of this cell death is unknown.
  • movement-related include shaking, rigidity, slowness of movement and difficulty with fine motor skills, walking, and gait.
  • thinking and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease, whereas depression is the most common psychiatric symptom.
  • Other symptoms include sensory, sleep and emotional problems.
  • PD is characterized by progressive motor impairment and neuroinflammation induced by microglia, the resident immune cells of the central nervous system (Aguzzi et al, Science, 339:156-61, 2013). Inflammatory mediators produced by dysfunctional microglia have been shown to induce neuronal cell death, which underlies the progressive impairment in cognitive and behavioral performance in neurodegenerative diseases (Czirr et al J Clin Invest, 122:1156-63, 2012). Nevertheless, specific signaling pathways that contribute to microglia-mediated inflammation remain elusive.
  • HD Huntington's disease
  • Frontotemporal degeneration is a disease that is closely related to AD in which progressive degeneration occurs in the frontal and temporal lobes of the brain. Gliosis and inflammatory activation of microglia have been documented in humans and animal models of FTD (Cagnin et al Annals of Neurol. 2004 6: 894-897; Yi et al. J. Exp. Med. 2010. 1:117-128). Patients with FTD experience a gradual decline in behavior and language with memory usually relatively preserved. As the disease progresses, it becomes increasingly difficult for afflicted subjects to organize activities, behave appropriately, and care for oneself. There are currently no treatments to slow or stop the progression of the disease.
  • Dementia with Lewy bodies is a type of dementia hat is related to PD.
  • the hallmark of this disease is the presence of alpha synuclein aggregates in brains of afflicted subjects. These patients experience PD-like symptoms including hunched posture, rigid muscles, a shuffling walk and trouble initiating movement as well as changes in reasoning and thinking, memory loss (but less significantly than AD).
  • Lewy bodies are also present in PD, these two diseases may be linked to the same underlying abnormalities in how the brain processes the protein alpha-synuclein.
  • microglia-related neuroinflammation is present in brains of subjects with DLB, although this pathological feature occurs more extensively (Iannaccone et al, Parkinsonism Relat. Disord. 2013 19: 47-52).
  • MND Motor neuron diseases
  • ALS Motor neuron diseases
  • progranulin can trigger inflammatory activation of microglia in an animal model of MND and genetic ablation of this pathway can delay disease progression (Philips et al J Neuropathol Exp Neurol. 2010 69:1191-200).
  • Demyelinating diseases such as Guillain-Barré syndrome and multiple sclerosis (MS) are degenerative disorders in which in which the myelin sheath of neurons is compromised. This damage impairs signal conductivity in the affected nerves, causing deficiency in sensation, movement, cognition, or other functions. There is no cure for these diseases. Its most well-known form is MS, a disease in which the cellular subsets of the immune system have been implicated. For instance, on-going demyelination is often associated with infiltration of T cells and macrophages from the circulation as well as inflammatory activation of microglia (Kutzelnigg et al. Handb. Clin. Neurol. 2014, 122:15-58).
  • the method should be effective and well tolerated.
  • WT wild-type animal
  • Veh animals treated with vehicle
  • MPTP-Ctrl animals treated with MPTP and 0.5% methylcellulose
  • MPTP-ZLN animals treated with MPTP and ZLN005
  • STZ-Ctrl animals treated with STZ and 0.5% methylcellulose
  • STZ-ZLN animals treated with STZ and ZLN005
  • 5XFAD-Ctrl AD transgenic animals treated with 0.5% methylcellulose
  • 5XFAD-ZLN AD transgenic animals treated with ZLN005
  • ALS-Ctrl ALS transgenic animals treated with 0.5% methylcellulose
  • ALS-ZLN ALS transgenic animals treated with ZLN005.
  • FF Ppargc1a LoxP/LoxP mice
  • Cre Ppargc1a LoxP/LoxP Cx3cr1 CreER mice.
  • FIG. 1 shows the survival rate as a percentage of Cre animals and FF animals within 30 hours after MPTP induction.
  • FIG. 2 shows that Ppargc1a activator ZLN005 increases expression of genes Pgc1a (Ppargc1a), Tfam, Nrf2, Ucp3, Ant, Sod1, Sod2 and upregulates tyrosine hydroxylase (Th).
  • FIG. 3 shows the Pgc1a (Ppargc1a) protein expression in microglia in animals treated with Veh, MPTP-Ctrl, and (MPTP-ZLN).
  • FIG. 4 shows the glucose transporter Slc2a1 levels and lactic acid levels in animals treated with Veh, MPTP-Ctrl, and MPTP-ZLN.
  • FIG. 5 shows immunohistochemical analysis of dopaminergic neurons in the substantia nigra of animals treated with Veh, MPTP-Ctrl, and MPTP-ZLN.
  • FIG. 6 shows TNF- ⁇ levels secreted by microglia in Cre animals and FF animals, treated with Veh, MPTP-Ctrl, and MPTP-ZLN.
  • FIG. 7 shows weights (g) of shredded nestlets by Cre animals and FF animals, treated with Veh, MPTP-Ctrl, and MPTP-ZLN.
  • FIG. 8 shows latency of fall (seconds) of Cre animals and FF animals, treated with Veh, MPTP-Ctrl, and MPTP-ZLN.
  • FIG. 9 shows relative expression level of several genes in animals treated with Veh, STZ-Ctrl, and STZ-ZLN.
  • FIG. 10 shows % of microglia that express TNF- ⁇ + (A), % ThioltrackerViolet hi (B), and % MitotrackerRed hi (C) in Veh, STZ-Ctrl, and STZ-ZLN.
  • FIG. 11 shows mean disease scores of STZ-Ctrl and STZ-ZLN mice.
  • FIG. 12 shows % of microglia that express IL1 (A) and TNF ⁇ (B), in WT, 5XFAD-Ctrl, and 5XFAD-ZLN.
  • FIG. 13 shows % of microglia that express Mitotracker Green hi (A), and % microglia that had taken up 2-NBDG (B), in WT, 5XFAD-Ctrl, and 5XFAD-ZLN.
  • FIG. 14 shows % blood monocytes over circulating immune cells in WT, 5XFAD-Ctrl, and 5XFAD-ZLN.
  • FIG. 15 shows nest building activities (g) in WT, 5XFAD-Ctrl, and 5XFAD-ZLN.
  • FIG. 16 shows % of brain perivascular macrophages that express iNOS, IL6, and TNF ⁇ in WT, ALS-Ctrl, and ALS-ZLN.
  • FIGS. 17A-B show latency of fall (seconds) of ALS transgenic animals, treated with 0.5% methylcellulose (Ctrl) or ZLN, at a constant speed ( FIG. 17A ) and at an accelerating speed ( FIG. 17B ) in a wheel-running test.
  • FIG. 18 shows % survival vs. time after 100 days in ALS transgenic animals treated with 0.5% methylcellulose (Ctrl) or ZLN005. Animals were treated 3 times a week starting at 5, 10, and 15 weeks of age.
  • FIG. 19 shows % of brain perivascular macrophages among total brain immune cells in the brain in WT, ALS-Ctrl, and ALS-ZLN.
  • FIG. 20 shows % of brain perivascular macrophages that have taken up a glucose analog 2-NBDG in WT, ALS-Ctrl, and ALS-ZLN.
  • FIGS. 21A and 21B show % of total monocytes and % of Ly6C+ inflammatory monocytes among circulating immune cells, in WT, ALS-Ctrl, and ALS-ZLN.
  • FIG. 21C shows the serum TNF- ⁇ levels in WT, ALS-Ctrl, and ALS-ZLN.
  • FIG. 22 shows latency of fall (seconds) of HD transgenic animals treated with 0.5% methylcellulose (Ctrl) or ZLN005.
  • FIGS. 23A and 23B show latency of fall (seconds) in FF and Cre mice.
  • FF Ppargc1a LoxP/LoxP mice on DLB transgenic background
  • Cre Ppargc1a LoxP/LoxP Cx3cr1 CreER mice mice on DLB transgenic background.
  • FIGS. 24A and 24B show latency of fall (seconds) of DLB transgenic animals treated with 0.5% methylcellulose (Ctrl) or ZLN005.
  • Inflammatory responses in the brain which can be demonstrated by changes in the properties of microglia, a cell type that is located only in the brain, are a common feature of human neurodegenerative diseases (Alzheimers Res Ther., 7(1):56. doi: 10.1186/s13195-015-0139-9, 2015).
  • Yong The Neuroscientist, 16:408-420, 2010
  • CNS central nervous system
  • microglia activation is a cause of this inflammatory response
  • microglia-mediated neuroinflammation is present in all neurodegenerative disorders.
  • Ppargc1a a pleotropic regulator of cellular metabolism in many cell types, is an important regulator of all neurodegenerative diseases, in which neuroinflammation is mediated by microglia.
  • the inventors have discovered a connection between Ppargc1 activation in microglia and its effect on the cognitive and motor functions of the whole organism.
  • the inventors have discovered that Ppargc1a expression is decreased in humans and animal models with neurodegenerative diseases.
  • the inventors have shown that Ppargc1a signaling in microglia is an important regulator of motor dysfunction and behavioral dysfunction in animal models and provided evidence that targeting Ppargc1a with its activator improves motor/behavior dysfunction in neurodegenerative diseases.
  • the present invention is directed to a method for treating neurodegenerative diseases.
  • the method comprises the step of administering an effective amount of a Ppargc1a activator to a subject suffering from a neurodegenerative disease.
  • Neurodegenerative diseases refers to diseases that occur as a result of neurodegenerative processes, i.e., progressive loss of structure or function of neurons and/or death of neurons. Neurodegenerative diseases are incurable and debilitating, and patients typically have problems with movement (ataxias) and/or mental functioning (dementias). Neurodegenerative diseases include ALS, AD, PD, HD, frontotemporal degeneration disease, dementia with Lewy bodies, motor neuron diseases, demyelinating diseases (such as Guillain-Barré syndrome and multiple sclerosis), prion disease, spinocerebellar ataxia, and spinal muscular atrophy.
  • ALS ALS
  • AD PD
  • HD frontotemporal degeneration disease
  • dementia with Lewy bodies dementia with Lewy bodies
  • motor neuron diseases demyelinating diseases (such as Guillain-Barré syndrome and multiple sclerosis)
  • demyelinating diseases such as Guillain-Barré syndrome and multiple sclerosis
  • prion disease spinocer
  • ZLN005 activation of the Ppargc1a pathway in microglia by ZLN005 can suppress microglia-mediated inflammatory responses.
  • Deletion of Ppargc1a specifically in microglia accelerates neuropathological development in transgenic animal models of PD (MPTP) and dementia with Lewy bodies (SNCA*A53T).
  • MPTP transgenic animal models of PD
  • AD 5XFAD and icv-STZ
  • HD R6/2
  • ALS SOD1*G93A
  • dementia with Lewy bodies SNCA*A53T
  • ZLN005 represents a treatment for all neurodegenerative disorders in which microglia-mediated neuroinflammation contributes to the disease development.
  • Circulating monocytes from the blood give rise to brain perivascular macrophages, which reside just outside the vascular basement membrane. They are the main antigen-presenting cells of the CNS, thus playing an important role in immune reactions involving the brain.
  • brain perivascular macrophages are the earliest macrophages from peripheral tissues that response to brain injuries. Their location at the interface between brain parenchyma and the vascular system and their continuous circulation in and out of blood vessels suits them ideally for this function.
  • the inventors have discovered that brain perivascular macrophages in ALS transgenic mice exhibited an inflammatory phenotype, evidenced by a significant increase in iNOS production.
  • ZLN005 By administering ZLN005 to these animals, iNOS production in the brain perivascular macrophages decreased and neuroinflammation was suppressed.
  • ALS transgenic mice treated with ZLN005 had improved motor skills compared with untreated ALS transgenic mice.
  • ALS transgenic mice exhibited hind limb paralysis at approximately 100 days and died shortly after.
  • ZLN005 the onset of hind limb paralysis was delayed and the survival rate increased.
  • the inventors have discovered that administering ZLN005 to the STZ-treated animal resulted in increased expression of genes involved in Ppargc1a signaling, mitochondrial metabolism, and anti-oxidative defense in the brain
  • the principal chemical constituent of the amyloid plaques and amyloid angiopathy characteristic of AD is an approximately 4.2 KD protein of ⁇ -amyloid peptide.
  • STZ-treated animals significantly increase the expression of ⁇ -amyloid peptide.
  • ZLN005 By administering ZLN005, the expression of genes involved in ⁇ -amyloid generation in the brains of STZ-treated animals was decreased to normal levels.
  • the inventors have shown that microglia in STZ-treated mice exhibited an inflammatory phenotype, evidenced by a significant increase in TNF- ⁇ production.
  • Administering ZLN005 to the STZ-treated mice resulted in suppression of TNF- ⁇ production in the microglia cells and suppression of the microglia-mediated neuroinflammation.
  • the inventors also discovered that ZLN005 modulated metabolic dysfunction in microglia induced by STZ, as evidenced by enhanced glycolysis, mitochondrial potential, and glutathione production in microglia isolated from STZ-treated animals and treated by ZLN005.
  • STZ-treated mice exhibit several signs and symptoms of behavioral dysfunction and systemic inflammation including bleeding from the nose, eyes, ears, paralysis of hands and feet (Arabpoor et al Adv Biomed Res, 1:50, 2012).
  • Administering ZLN005 to the STZ-treated mice resulted in a significant reduction in the disease severity.
  • the inventors have generated microglia specific knockout of Ppargc1a, in which Ppargc1a signaling is absent in these cells and not in other cells of the brain such as neurons.
  • PD was induced with MPTP in wild-type and microglia specific knockout animals, the knockout animals had significantly more severe motor impairment, indicating that Ppargc1a signaling in microglia regulates behavioral dysfunction.
  • microglia in MPTP-treated mice exhibited an inflammatory phenotype, evidenced by a significant increase in TNF- ⁇ production and a decrease in mitochondrial biogenesis.
  • Administering ZLN005 to the MPTP-treated mice resulted in decreased TNF- ⁇ production in the microglia cells and suppression of microglia-mediated neuroinflammation.
  • MPTP-treated mice exhibit profound loss of fine motor skills and behavioral dysfunctions.
  • the inventors have shown that by administering ZLN005 to the MPTP-treated mice, the motor skills of those mice were improved.
  • Targeting Ppargc1a with its activator, ZLN005 ameliorates motor dysfunction in Huntington's disease (HD).
  • the inventors have provided evidence that targeting Ppargc1a with ZLN005 improved motor skills in HD transgenic mice.
  • the inventors have shown that HD transgenic mice treated with ZLN005 exhibited improved motor skills, as indicated by increases in their latency to fall, compared with untreated HD transgenic mice.
  • Ppargc1a with its activator ZLN005 ameliorates motor dysfunction in dementia with Lewy bodies.
  • the inventors have shown that microglia-specific deletion in transgenic DLB animals caused further deterioration of motor function in the animals.
  • the inventors have also demonstrated that Ppargc1a activator, ZLN005, improved motor skills in transgenic animals.
  • Frontotemporal degeneration also called frontotemporal dementia (FTD) is a disease that is closely related to ALS in which progressive degeneration occurs in the frontal and temporal lobes of the brain.
  • FTD frontotemporal dementia
  • ZLN005 By suppressing microglia-mediated inflammation, ZLN005 improves motor skills in FTD transgenic mice and increases their survival rate.
  • Motor neuron diseases are neurodegenerative disorders, similar to ALS, that selectively affect motor neurons.
  • Microglia-mediated inflammation is a key factor for development factor for motor neuron diseases. By suppressing microglia-mediated inflammation, ZLN005 slows down and halts disease development.
  • ZLN005 is effective in treating demyelinating diseases by reducing the inflammatory activation of microglia, which might be more susceptible to inflammatory stimuli in demyelinating diseases such as multiple sclerosis. By suppressing metabolic dysregulation and subsequent inflammatory transformation of microglia, ZLN005 promotes myelin repair and regeneration.
  • the present invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and an active compound of 2-(4-tert-butylphenyl)-1H-benzimidazole, 2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole (ZLN005), or a pharmaceutically acceptable salt, or a solvate thereof.
  • the active compound or its pharmaceutically acceptable salt or solvate in the pharmaceutical compositions in general is in an amount of about 0.01-20% (w/w) for a topical formulation; about 0.1-5% for an injectable formulation, 0.1-5% for a patch formulation, about 1-90% for a tablet formulation, and 1-100% for a capsule formulation.
  • the pharmaceutical composition can be in a dosage form such as tablets, capsules, granules, fine granules, powders, syrups, suppositories, injectable solutions, patches, or the like.
  • the pharmaceutical composition can be an aerosol suspension of respirable particles comprising the active compound, which the subject inhales.
  • the respirable particles can be liquid or solid, with a particle size sufficiently small to pass through the mouth and larynx upon inhalation. In general, particles having a size of about 1 to 10 microns, preferably 1-5 microns, are considered respirable.
  • the active compound is incorporated into any acceptable carrier, including creams, gels, lotions or other types of suspensions that can stabilize the active compound and deliver it to the affected area by topical applications.
  • any acceptable carrier including creams, gels, lotions or other types of suspensions that can stabilize the active compound and deliver it to the affected area by topical applications.
  • the above pharmaceutical composition can be prepared by conventional methods.
  • Pharmaceutically acceptable carriers which are inactive ingredients, can be selected by those skilled in the art using conventional criteria.
  • Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous based solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments.
  • the pharmaceutically acceptable carriers may also contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxide, phosphate, citrate, acetate, borate; and trolamine; antioxidants such as salts, acids and/or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, and ascorbyl palmitate; surfactants such as lecithin, phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositiol; poloxa
  • Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives, these include, but are not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use.
  • preservatives include, but are not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use.
  • a tablet formulation or a capsule formulation of the active compound may contain other excipients that have no bioactivity and no reaction with the active compound.
  • Excipients of a tablet or a capsule may include fillers, binders, lubricants and glidants, disintegrators, wetting agents, and release rate modifiers. Binders promote the adhesion of particles of the formulation and are important for a tablet formulation.
  • excipients of a tablet or a capsule include, but not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone.
  • a tablet formulation may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and/or titanium dioxide.
  • a capsule formulation may contain inactive ingredients such as gelatin, magnesium stearate, and/or titanium dioxide.
  • a patch formulation of the active compound may comprise some inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water.
  • a patch formulation may also contain skin permeability enhancer such as lactate esters (e.g., lauryl lactate) or diethylene glycol monoethyl ether.
  • Topical formulations including the active compound can be in a form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, and suspension.
  • the inactive ingredients in the topical formulations for example include, but not limited to, diethylene glycol monoethyl ether (emollient/permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modifier), caprylic/capric triglyceride, (emollient), octisalate, (emollient/UV filter), silicone fluid (emollient/diluent), squalene (emollient), sunflower oil (emollient), and silicone dioxide (thickening agent).
  • diethylene glycol monoethyl ether emollient/permeation enhancer
  • DMSO solubility enhancer
  • silicone elastomer rheology/texture modifier
  • caprylic/capric triglyceride e
  • the present invention is directed to a method of treating neurodegenerative diseases.
  • the method comprises the step of administering to a subject suffering from a neurodegenerative disease an effective amount of 2-(4-tert-butylphenyl)-1H-benzimidazole, 2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole, for treating the neurodegenerative disease.
  • An effective amount is the amount effective to treat the neurodegenerative disease by ameliorating the pathological condition or reducing the symptoms of the disease.
  • the neurodegenerative disease is ALS and the method reduces or alleviates motor dysfunction or behavioral dysfunction in an ALS patient.
  • the method improves early symptoms such as difficulty in walking or doing normal daily activities; weakness in legs, feet, ankles, or hand; tripping or clumsiness; slurring of speech or trouble swallowing; and muscle cramps and twitching in the arms, shoulders and tongue.
  • the method may also improve later symptoms such as difficulty in breathing.
  • the method improves survival rate and length of survival.
  • the neurodegenerative disease is AD and the method reduces or alleviates the disease symptoms and improves the cognitive and motor functions. For example, the method improves confusion, irritability, aggression, mood swings, trouble with language, and/or long-term memory loss in a patient. The method may also slow down the disease progression.
  • the neurodegenerative disease is PD and the method reduces or alleviates motor dysfunction or behavioral dysfunction in a patient.
  • the method improves movement-related symptoms such as shaking, rigidity, slowness of movement, and difficulty with fine motor skills, walking, and gait.
  • the neurodegenerative disease is HD and the method reduces or alleviates motor dysfunction in a patient.
  • the method improves involuntary and/or voluntary movement-related symptoms such as involuntary jerking or writhing movements (chorea); muscle problems (e.g., rigidity or muscle contracture (dystonia)); slow or abnormal eye movements; impaired gait, posture and balance; difficulty with the physical production of speech or swallowing.
  • the neurodegenerative disease is dementia with Lewy bodies (DLB) and the method reduces or alleviates motor dysfunction and cognitive decline in a patient.
  • the method improves PD-like symptoms such as motor coordination, difficulties with walking and swallowing, inability to maintain normal postures, rigidity as well as loss of memory and decline in thinking and reasoning.
  • the method may also halt or slow down disease progression.
  • the neurodegenerative disease is frontotemporal degeneration (FTD) and the method reduces or alleviates the disease symptoms that are associated with language skills and social interactions.
  • FTD frontotemporal degeneration
  • the method improves abilities to speak coherently, to organize thoughts and daily activities, to interact normally in social settings and alleviates symptoms of disinhibition, loss of sympathy and empathy, lack of executive control, hyperorality, and apathy.
  • the method may also halt or slow down disease progression.
  • the neurodegenerative disease is a motor neuron disease (MND) and the method reduces or alleviates motor dysfunction as well as improves survival rate and length of survival of patients with these diseases.
  • MND motor neuron disease
  • the method improves movement-related symptoms such as troubles with walking, maintaining normal gait, controlling balance, difficulties with fine motor coordination, slowness of movement, swallowing, and breathing.
  • the neurodegenerative disease is a demyelinating disease such as Guillain-Barré syndrome or multiple sclerosis (MS) and the method reduces or alleviates behavioral dysfunction and cognitive impairment in patients with these diseases.
  • the method improves early symptoms such as blurred vision, tingling sensation, numbness and weakness in limbs, lack of coordination.
  • the method may also improve advanced symptoms such as difficulty in walking, tremors, muscle spasms, paralysis, troubling articulating thoughts and speaking.
  • the method may also improve survival rate and length of survival.
  • the pharmaceutical composition of the present invention can be applied by systemic administration or local administration.
  • Systemic administration includes, but is not limited to oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal), and inhaled administration.
  • parenteral such as intravenous, intramuscular, subcutaneous or rectal
  • inhaled administration In systemic administration, the active compound first reaches plasma and then distributes into target tissues.
  • Oral administration is a preferred route of administration for the present invention.
  • Local administration includes topical administration.
  • Dosing of the composition can vary based on the extent of the injury and each patient's individual response.
  • plasma concentrations of the active compound delivered can vary; but are generally 1 ⁇ 10 ⁇ 10 -1 ⁇ 10 ⁇ 4 moles/liter, and preferably 1 ⁇ 10 ⁇ 8 -1 ⁇ 10 ⁇ 5 moles/liter.
  • the pharmaceutical composition is administrated orally to a subject.
  • the dosage for oral administration is generally 0.1-100, 0.1-20, or 1-50 mg/kg/day, depending on the subject's age and condition.
  • the dosage for oral administration is 0.1-10, 0.5-10, 1-10, 1-5, or 5-50 mg/kg/day for a human subject.
  • the active compound can be applied orally to a human subject at 1-100, 10-50, 20-1000, 20-500, 100-800 sage, or 200-600 mg/dosage, 1-4 times a day, depends on the patient's age and condition.
  • the pharmaceutical composition is administrated intravenously to a subject.
  • the dosage for intravenous bolus injection or intravenous infusion is generally 0.03 to 5 or 0.03 to 1 mg/kg/day.
  • the pharmaceutical composition is administrated subcutaneously to the subject.
  • the dosage for subcutaneous administration is generally 0.3-20, 0.3-3, or 0.1-1 mg/kg/day.
  • the composition is applied topically to an area and rubbed into it.
  • the composition is topically applied at least 1 or 2 times a day, or 3 to 4 times per day, depending on the medical issue and the disease pathology.
  • the topical composition comprises about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5% (w/w) of the active compound.
  • 0.2-10 mL of the topical composition is applied to the individual per dose.
  • the active compound passes through skin and is delivered to the site of discomfort.
  • the present invention is useful in treating a mammal subject, such as humans, horses, dogs and cats.
  • the present invention is particularly useful in treating humans.
  • mice with microglia-specific deletion of Ppargc1a were generated by crossing mice harboring the foxed allele of Ppargc1a (Ppargc1a LoxP/LoxP ) with those expressing Tamoxifen inducible Cre recombinase under the control of Cx3cr1 promoter (Cx3cr1 CreER ).
  • MPTP (20 mg/kg) was administered intraperitoneally in sterile PBS 4 times at 2-hour intervals on day 1. Control animals received a similar volume of PBS. After MPTP induction, 7 out of 12 Cre animals died within 30 hours, while 3 out of 16 FF animals died within 30 hours. The results are shown in FIG. 1 . Log-rank test was used for statistical analysis. The results show that Ppargc1a deletion in microglia accelerates MPTP-induced mortality.
  • Ppargc1a an inducer of mitochondrial biogenesis, is widely expressed in cells throughout the body.
  • Ppargc1a activator ZLN005 25 mg/kg, Sigma was administered orally once a day starting 30 minutes after MPTP administration on Day 1 (when animals exhibited PD-like symptoms) for 3 consecutive days in 0.5% methylcellulose (Sigma).
  • mice were sacrificed on Day 4, 24 hours after the third oral dosage of ZLN005 and PBS-perfused brain tissues were processed for microglia isolation and flow cytometry analysis of glucose metabolism in microglia.
  • Microglia were sorted by flow cytometry and subjected to lactic acid production assays ex vivo (Cayman Chem) for glycolysis measurement.
  • Y-axis represents Slc2a1 expression in median fluorescence units (MFI, A) and lactic acid production in micromolar units (mM, B).
  • MFI median fluorescence units
  • mM micromolar units
  • the results show that microglia in MPTP-treated mice exhibited a glycolytic activation phenotype, measured by increases in glucose transporter Slc2a1 expression (A) and lactic acid production (B), in non-treated MPT-intoxicated animals when compared with Veh mice.
  • the results also show that by administering ZLN005 to MPTP-treated animals, glucose transporter expression and lactic acid production in microglia of these treated animals decreased, and thus their metabolic dysfunction was corrected.
  • ANOVA was used for statistical analyses.
  • Ppargc1a activator ZLN005 25 mg/kg, Sigma was administered orally once a day starting 30 minutes after MPTP administration on Day 1 for 7 consecutive days in 0.5% methylcellulose (Sigma).
  • animals were sacrificed on Day 8, 24 hours after the 7th oral dosage of ZLN005, and paraformaldehyde-perfused brain tissues were processed for immunohistochemical analysis of dopaminergic neurons in the substantia nigra.
  • the brown staining represents tyrosine hydroxylase expression in dopaminergic neurons of the substantia nigra.
  • the results show that MPTP administration led to a depletion of these neurons, which was reversed by treatment with ZLN005.
  • Example 6 Suppresses TNF- ⁇ Production in a Microglial Ppargc1a Dependent Manner
  • results are summarized in FIG. 6 .
  • the results show that MPTP administration induced TNF- ⁇ secretion by microglia in FF animals and this induction of TNF- ⁇ production was significantly higher in Cre animals.
  • ZLN005 suppressed TNF- ⁇ production in microglia isolated from MPTP-treated FF animals but failed to exert its anti-inflammatory effects on microglia isolated from MPTP-treated Cre animals, which had Cre-mediated deletion of Ppargc1a in Cx3cr1 expressing microglia.
  • These results indicate that ZLN005 suppresses expression of the inflammatory cytokine TNF- ⁇ in microglia via its activation of microglia specific Ppargc1a. Unpaired t-tests were used for statistical analyses.
  • Example 7 Improves Fine Motor Skills in a Microglial Ppargc1a Dependent Manner
  • Example 8 Improves Motor Coordination in a Microglial Ppargc1a Dependent Manner
  • mice with microglia-specific deletion of Ppargc1a were generated as described in Example 1. At 7 weeks of age, these animals were subjected to 1.5 weeks of training on a treadmill at a constant speed 10 rpm (rotations per minute) and then 1.5 weeks of training at an accelerating speed from 5-15 rpm. After the training period at 10 weeks of age, animals were treated with MPTP and tested for motor performance at an accelerating speed from 5-15 rpm.
  • FIG. 8 shows that MPTP treatment impaired wheel running time in FF animals and ZLN005 treated mice performed significantly better than vehicle treated mice.
  • the ability of ZLN005 to improve wheel-running skills is not present in Cre animals, which had Cre-mediated deletion of Ppargc1a in Cx3cr1 expressing microglia.
  • Veh animals treated with artificial cerebrospinal fluid as vehicle in STZ model
  • STZ-Ctrl animals treated with STZ and 0.5% methylcellulose
  • STZ-ZLN animals treated with STZ and ZLN005
  • WT wild-type animals
  • 5XFAD-Ctrl transgenic AD animals treated with 0.5% methylcellulose
  • 5XFAD-ZLN transgenic AD animals treated with ZLN005.
  • Ppargc1a which is an activator of mitochondrial biogenesis, is widely expressed in cells throughout the body.
  • Ppargc1a activator ZLN005 25 mg/kg, Sigma
  • ZLN005 25 mg/kg, Sigma
  • ZLN005 0.5% methylcellulose
  • mice were sacrificed on Day 4, and PBS-perfused brain tissues were processed for RNA isolation, cDNA synthesis and real-time quantitative PCR (Invitrogen).
  • results are summarized in FIG. 9 .
  • Example 10 Ppargc1a Activator ZLN005 Suppresses TNF- ⁇ Production and Metabolic Abnormalities in Microglia in the Acute STZ Model of AD
  • Ppargc1a activator ZLN005 25 mg/kg, Sigma was administered orally once in 0.5% methylcellulose (Sigma) immediately before the first dose of STZ on Day 1. Treatment with ZLN005 was continued on a daily schedule until Day 7.
  • microglia analysis animals were sacrificed on Day 7, and PBS-perfused brain tissues were digested with Collagenase IV and processed for flow cytometry. Microglia were phenotyped with antibodies directed against mouse TNF- ⁇ (Biolegend) and metabolic dyes ThioltrackerViolet, and MitotrackerRed (Invitrogen) for flow cytometric acquisition (LSRII, BD) and analysis (FlowJo). The results are summarized in FIG. 10 .
  • Ppargc1a activator ZLN005 25 mg/kg, Sigma was administered orally once on Day 1 in 0.5% methylcellulose (Sigma) immediately before the first dose of STZ. Treatment with ZLN005 continued on a daily schedule until Day 4, when the animals were evaluated.
  • STZ-ZLN mice 33% of the animals were active, 66% showed evidence of lethargy, and none were paralyzed.
  • STZ-Ctrl mice only 10% were active, 70% were lethargic, and 20% of these animals had hind limb paralysis.
  • Veh animals receiving intracerebral artificial cerebrospinal fluid exhibited normal behavior.
  • the inventors designed a disease scoring system based on evidence of tissue inflammation: with scores of 1 (mild inflammation, increased vascularization/bleeding of internal organs), 2 (moderate inflammation, severe vascularization/bleeding of internal organs), and 3 (severe inflammation, intestinal or stomach swelling).
  • the disease scoring system is also based on physical activity with scores of 1 (lethargic, general poverty of movements with signs of lethargy), 2 (inactive, lack of movement for more than 15 consecutive seconds), and 3 (paralysis of either front or hind limbs).
  • the disease score presented in the example is the total score of the two scoring systems.
  • the mean disease scores of animals on day 4 in STZ-Ctrl and STZ-ZLN are shown in FIG. 11 .
  • STZ-ZLN mice had a significantly lower mean disease score (1.6) compared to STZ-Ctrl (2.5), indicating that the disease severity was improved by the ZLN005 treatment.
  • Animals receiving intracerebral artificial cerebrospinal fluid behaved normally and had a mean score of 0. Unpaired t-test was used for statistical analysis.
  • 5XFAD transgenic mice which are model of familial AD, were purchased from Jackson Laboratories (Oakley et al J Neurosci. 26:10129-40, 2006). These animals overexpress both mutant human APP(695) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer's Disease (FAD) mutations and human PS1 harboring two FAD mutations, M146L and L286V. These transgenic mice rapidly recapitulate major features of amyloid pathology in AD by 8-10 weeks of age. Microglia abnormalities and neuroinflammation are also pronounced within this time window. Subsequently, neurodegeneration and behavioral dysfunction that mimic cognitive and psychiatric symptoms of human AD begin and are pronounced by 4-5 months of age.
  • AD transgenic animals were orally treated 3 times a week for 4 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 3 weeks of age.
  • Y-axis represents % of microglia that express IL1 (A) and TNF ⁇ (B).
  • the results show that microglia in AD transgenic mice exhibited an inflammatory phenotype, evidenced by a significant increase in IL1 production in 5XFAD-Ctrl when compared with WT mice.
  • the results also show that by administering ZLN005 to AD transgenic animals, IL1 and TNF ⁇ production in microglia of these treated animals decreased and thus neuroinflammation was suppressed. ANOVA was used for statistical analyses.
  • AD transgenic animals were orally treated 3 times a week for 4 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 3 weeks of age.
  • PBS-perfused brain tissues of sacrificed animals were digested with Collagenase IV and processed for flow cytometry.
  • Brain microglia were phenotyped with 2-NBDG and MitotrackerGreen (Invitrogen) for flow cytometric acquisition (LSRII, BD) and analysis (FlowJo).
  • Y-axis represents % of microglia that highly expressed MitotrackerGreen (A) and had taken up 2-NBDG (B).
  • Mitochondrial respiration and glycolysis are two key energy generating pathways in living cells.
  • immune cells like microglia, inflammatory transformation is associated with upregulation of glucose utilization and depression of mitochondrial biogenesis and function.
  • the results show that microglia in 5XFAD-Ctrl exhibited a decrease in mitochondrial mass, measured by Mitotracker Green (A), and exhibited a glycolytic activation phenotype, evidenced by a significant increase in glucose uptake, measured by 2-NBDG incorporation (B), when compared with WT animals.
  • AD transgenic animals were orally treated 3 times a week for 4 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 3 weeks of age.
  • Y-axis represents % of total circulating monocytes among circulating immune cells.
  • the results show that the percentage of blood monocytes was increased in 5XFAD-Ctrl when compared with WT mice.
  • the results also show that by administering ZLN005 to AD transgenic animals, the percentage of monocytes decreased. ANOVA was used for statistical analysis.
  • nest-building skill is one of the most reliable measurements of motor function.
  • AD transgenic animals were orally treated 3 times a week for 4 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 3 weeks of age.
  • ZLN005 Sigma
  • animals were given cotton pads and the amount of cotton that was shredded over a 24-hour period was measured.
  • the nest building activities (g) are shown in FIG. 15 .
  • WT wild-type animals
  • ALS-Ctrl transgenic ALS animals treated with 0.5% methylcellulose
  • ALS-ZLN transgenic ALS animals treated with ZLN005.
  • ALS transgenic animals were purchased from Jackson Laboratories. These animals express the G93A mutation in the gene SOD1 which has been implicated as the cause of the disease in a subset of human subjects with familial ALS. The animals exhibit hind limb paralysis, a classical symptom of ALS, upon 100-110 days of age and rapidly succumb. These animals represent a gold standard model for therapeutic discovery in the field of ALS research.
  • ALS transgenic animals were orally treated 3 times a week for 8 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 5 weeks of age.
  • PBS-perfused brain tissues of sacrificed animals were digested with Collagenase IV and processed for flow cytometry.
  • Brain perivascular macrophages were phenotyped with antibodies directed against mouse iNOS, IL6, and TNF ⁇ (Biolegend) for flow cytometric acquisition (LSRII, BD) and analysis (FlowJo).
  • Y-axis represents % of brain perivascular macrophages that express iNOS (A), IL6 (B) and TNF ⁇ (C).
  • the results show that brain perivascular macrophages in ALS transgenic mice exhibit an inflammatory phenotype, evidenced by a significant increase in iNOS, IL6, and TNF ⁇ production in ALS-Ctrl mice when compared with WT animals.
  • the results also show that by administering ZLN005 to ALS transgenic animals, iNOS production in the brain perivascular macrophages of these treated animals decreased and thus neuroinflammation was suppressed.
  • IL6 and TNF ⁇ production in the brain perivascular macrophages of ZLN005 treated animals were also suppressed, although these differences did not reach statistical significance. ANOVA was used for statistical analyses.
  • ALS transgenic mice were orally treated 3 times a week for 4 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 9 weeks of age.
  • a wheel-running test was performed similarly to that described in Example 8.
  • the animals started training at 13 weeks of age for 1.5 weeks of training on a treadmill at a constant speed of 10 rpm and then for 1.5 weeks of training at an accelerating speed from 5-15 rpm. After the training period at 14.5 and 16 weeks of age, animals were tested for motor performance at a constant speed and at an accelerating speed, respectively.
  • the results are shown in FIGS. 17A-17B .
  • ALS transgenic mice were orally treated 3 times a week with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 5, 10 and 15 weeks of age.
  • FIG. 18 demonstrate that ALS-ZLN at 5 or 10 weeks of age significantly increased survival (mean survival of 131-132 days) in comparison to ALS-Ctrl (mean survival of 119 days); p-values ⁇ 0.05. Log-rank test was used for statistical analysis.
  • ALS transgenic animals were orally treated 3 times a week for 8 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 5 weeks of age.
  • PBS-perfused brain tissues of sacrificed animals were digested with Collagenase IV and processed for flow cytometry.
  • Y-axis represents % of brain perivascular macrophages among total brain immune cells in the brain.
  • the results show an increase in the percentage of brain perivascular macrophages in ALS-Ctrl mice when compared with WT mice.
  • the results also show that by administering ZLN005 to ALS transgenic animals, the percentage of the brain perivascular macrophages of these treated animals decreased. ANOVA was used for statistical analysis.
  • Example 20 Ppargc1a Activator ZLN005 Suppresses Glycolytic Activation in Brain Perivascular Macrophages in ALS Transgenic Animals
  • ALS transgenic animals were orally treated 3 times a week for 8 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 5 weeks of age.
  • ALS-ZLN ALS-ZLN
  • PBS-perfused brain tissues of sacrificed animals were digested with Collagenase IV and processed for flow cytometry.
  • Brain perivascular macrophages were stained with 2-NBDG, the fluorescent glucose analog, to measure glucose uptake for flow cytometric acquisition (LSRII, BD) and analysis (FlowJo).
  • Y-axis represents % of brain perivascular macrophages that have taken up the glucose analog, 2-NBDG.
  • the results show that brain perivascular macrophages in ALS transgenic mice exhibited a glycolytic phenotype, evidenced by a significant increase in 2-NBDG uptake in ALS-Ctrl mice when compared with WT mice.
  • the results also show that by administering ZLN005 to ALS transgenic animals, glucose uptake in the brain perivascular macrophages of these ALS-ZLN animals decreased and thus glycolytic activation and metabolic dysfunction in brain perivascular macrophages in ALS transgenic animals were suppressed.
  • ANOVA was used for statistical analysis.
  • ALS transgenic animals were orally treated 3 times a week for 8 weeks with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 5 weeks of age.
  • the Y-axis in FIGS. 21A and 21B represents % of total monocytes and % of Ly6C+ inflammatory monocytes among circulating immune cells.
  • the results show that monocytes, especially the Ly6C+ subset, were increased in ALS-Ctrl n compared with wild-type mice.
  • the results also show that by administering ZLN005 to ALS transgenic animals, the percentage of these cells in treated animals decreased and thus systemic inflammation was suppressed.
  • FIG. 21C shows that serum levels of TNF- ⁇ measured by ELISA in ALS transgenic animals were significantly suppressed by ZLN005 treatment Unpaired t-tests and ANOVA were used for statistical analyses.
  • Ctrl transgenic HD animals treated with 0.5% methylcellulose
  • ZLN transgenic HD animals treated with ZLN005.
  • HD transgenic animals (R6/2) were purchased from Jackson Laboratories. The animals exhibit symptoms of HD such as hind limb paralysis, muscle wasting, and impaired motor coordination, upon 8-10 weeks of age and rapidly succumb.
  • Ctrl DLB transgenic animals treated with 0.5% methylcellulose
  • ZLN DLB transgenic animals treated with ZLN005.
  • FF Ppargc1a LoxP/LoxP mice on DLB transgenic background
  • Cre Ppargc1a LoxP/LoxP Cx3cr1 CreER mice on DLB transgenic background.
  • SNCA*A53T transgenic mice an animal model in which the mutated form of human alpha synuclein is overexpressed, were generated to study pathological mechanisms in PD and DLB (Lee et al, Proc Natl Acad Sci USA. 2002, 13:8968-8970), These animals exhibit accumulation of pathogenic Lewy bodies upon aging, resulting in progressive motor dysfunction and eventual death.
  • mice with tnicroglia-specific deletion of Ppargc1a were generated as described in Example 1. Furthermore, these animals were bred with SNCA*A53T animals to generate mice with microglia-specific deletion of Ppargc1a on DLB genetic background. After tamoxifen treatment to induce deletion of Ppargc1a in microglia, animals were rested for 5 weeks before being subjected to treadmill training. At 8 weeks of age, these animals were subjected to 1.5 weeks of training on a treadmill at a constant speed 10 rpm (rotations per minute) and then 1.5 weeks of training at an accelerating speed from 5-15 rpm as described in Example 8. After the training period at 9.5 and 11 weeks of age, animals were tested for motor performance at a constant speed and at an accelerating speed, respectively.
  • FIGS. 23A-23B are representative of two independent experiments of one animal per genotype with similar outcomes. FF animals exhibited significantly longer latency to falls (average of two running trials) than Cre animals, which had Cre-mediated deletion of Ppargc1a in Cx3cr1 expressing microglia, at both constant speed of 10 rpm and accelerating speed of 5-15 rpm. These results show that microglia-specific Ppargc1a protects against motor dysfunction in this transgenic model of DLB.
  • DLB transgenic animals were purchased from Jackson Laboratories and were orally treated 3 times a week with 0.5% methylcellulose (Ctrl) or ZLN005 (ZLN) at 25 mg/kg in vehicle, starting at 8 weeks of age for 12 weeks.
  • mice were subjected to 1.5 weeks of training on a treadmill at a constant speed 10 rpm and then 1.5 weeks of training at an accelerating speed from 5-15 rpm, similar to those described in Example 8.
  • animals were tested for motor performance at a constant speed and at an accelerating speed, respectively.
  • DLB-ZLN mice performed significantly better than DLB-Ctrl mice (194.0 seconds vs. 134.5 seconds, p value ⁇ 0.05).
  • motor dysfunction of DLB mice was alleviated by ZLN005 treatment. Unpaired t-tests were used for statistical analyses.
  • TARDBP*A315T transgenic mice have been generated as an animal model to study ALS and FTD. These animals overexpress a mutant form of the DNA binding protein TARDBP, whose cytoplasmic inclusions are present in the brains of subjects with ALS and FTD (Barmada et al Nat Chem. Biol., 10:677-685, 2014).
  • Ke et al Short-term Suppression of A315T Mutant Human TDP-43 Expression Improves Functional Deficits in a Novel Inducible Transgenic Mouse Model of FTLD-TDP and ALS, Acta Neuropathol. 2015 Oct.
  • FTD transgenic animals are purchased from Jackson Laboratories and are orally treated 3 times a week with 0.5% methylcellulose (FTD-Ctrl) or ZLN005 (FTD-ZLN) at 25 mg/kg in vehicle, starting at 6 weeks of age. Subsequently, at 10 weeks of age, these animals are subjected to 1.5 weeks of training on a treadmill at a constant speed 10 rpm and then 1.5 weeks of training at an accelerating speed from 5-15 rpm. After the training period at 11.5 and 13 weeks of age, animals are tested for motor performance at a constant speed and at an accelerating speed, respectively. Finally, they are monitored for survival analysis.
  • FTD-Ctrl 0.5% methylcellulose
  • FTD-ZLN ZLN005

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CN114984008A (zh) * 2022-06-10 2022-09-02 南通大学 2-（4-叔丁基苯基）-1h-苯并咪唑在制备治疗帕金森病的药物制剂中的应用
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US10966962B2 (en) 2021-04-06
US10583125B2 (en) 2020-03-10
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US20200163941A1 (en) 2020-05-28
US20170304269A1 (en) 2017-10-26

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